The long-term objective of this project is the determination of the sequence of molecular events during duplication of E. coli, and the biochemical mechanisms which control the initiation and completion of each of the major steps in the sequence. A major objective for the current project is the determination of the molecular mechanisms which control the initiation of DNA replication. Four specific projects will be undertaken. First, the number of F' plasmids per cell, and the timing of replication of the plasmids during the division cycle, will be determined by transferring synchronous cultures containing an F' plasmid which is temperature-sensitive for replication to non-permissive temperature at various times in the cell cycle. Measurements of the segregation of the non-replicating plasmids into daughter cells will enable determination of the number of plasmids present at the same time of the temperature shift. Second, an attempt will be made to transfer gene segments near the origin of chromosome replication (which may be involved in determining the cellular age distribution at division) between various substrains of E. coli in an effort to determine the functions of these genes and to map their loci. Third, the mechanism which controls the timing of initiation of chromosome replication will be analyzed for performing temperature shifts in synchronous cultures of E. coli B/r dnaC. The relationship between cell division and initiation of chromosome replication in the mutant at the non-permissive temperature will be analyzed. Fourth, a newly isolated recA mutant of E. coli B/r will be used to investigate the coupling between chromosome replication and cell division during thymine starvation, and to generate F' plasmids possessing the origin of chromosome replication. BIBLIOGRAPHIC REFERENCES: Pierucci, O. and Helmstetter, C.E. Chromosome Segregation in E. coli B/r at Various Growth Rates. J. Bacteriol. 128, 708, 1976. Ron, E.C., Grossman, N. and Helmstetter, C.E. Control of Cell Division in E. coli - Effect of Amino Acid Starvation. J. Bacteriol., 1977.